1. Technical Field
Embodiments of the presently disclosed invention relate generally to immunomodulatory polypeptides, compositions comprising such polypeptides, and methods of using the same. More specifically, the present embodiments relate to short immunomodulatory polypeptides that specifically bind with low affinity to the human and murine toll-like receptors TLR2 and TLR4 such that they advantageously interfere with TLR2-and/or TLR4-mediated signaling.
2. Description of the Related Art
Sepsis, Inflammation and Immunity
The interaction between a host organism, such as a human, a mammal or another vertebrate animal, and a microbial pathogen such as a bacterium, virus, fungus or parasite, is complex and depends on genetic and environmental factors (e.g., nutrition, health, temperature); the interplay of these components may play a defining role in clinical outcome. One potential outcome of the host-pathogen interaction may be a subclinical host protective response by which the host eradicates the infection without any clinical evidence of infection, or, the host and pathogen may engage in a battle that produces clinical symptoms but during which the host maintains control of the pathogen and eventually eradicates it. Alternatively, in sepsis a more intense battle may ensue as a consequence of which the pathogen and/or the host's own inflammatory response eventually overwhelms the host, resulting in death of the host.
Sepsis is a severe, systemic inflammatory condition that occurs in about 750,000 to 900,000 people each year in the United States, and approximately one-third of sepsis patients die (Kellum et al., 2007 Arch Intern Med 167(15):1655-1663). Published studies have concluded that severe sepsis is a common, expensive, and frequently fatal condition that deserves more universal attention (Decker, 2004 J. Clin Invest 113(10): 1387-1389). Human sepsis may be identified in a patient when the patient meets at least two of the four criteria for Systemic Inflammatory Response Syndrome (SIRS) and also demonstrates features of MODS (multiple organ dysfunction syndrome) (Remick, 2007 Am. J. Pathol. 170(5):1435-1444). The SIRS criteria include (1) body temperature >38° C. or <36° C., (2) heart rate >90 beats per minute (bpm), (3) respiratory rate >20 breaths per minute or arterial CO2<32 mm Hg, and (4) circulating white blood cell (WBC) count >12,000/mm3 or <4000/mm3 or >10% immature forms (Remick 2007).
The innate immune system depends on the interplay between the pathogen associated molecular patterns (PAMPs) expressed by bacterial, viral, fungal and parasitic agents, and the pattern recognition receptors (PRRs) expressed by the cells of the host innate immune system, including macrophages, monocytes, neutrophils, and dendritic cells. Association of the immune cell surface receptor (PRR) and the pathogen cell surface ligand (PAMP) promotes relatively prompt phagocytosis of the pathogens by innate immune cells and also triggers the expression by these host cells of the CD80 and CD86 surface proteins, which are important in recruiting the activation of the adaptive (acquired) immune system, over time, to generate an antigen-specific immune response.
Toll-like receptors (TLRs) are the PRRs of primary importance in higher mammals: in the human innate immune system there are at least ten different TLR polypeptides, while in mice there are at least 11 different TLRs (Rich, T., Toll and Toll-Like Receptors: An Immunologic Perspective. 2010 Kluwer Academic/Plenum Publishers, Dordrecht, N L; see also, e.g., Gorden et al., 2005 J. Immunol. 174:1259 and references cited therein). These type I integral membrane proteins associate with adaptor proteins and, when engaged by appropriate ligands, mediate signal transduction that results in activation of downstream transcription factors (e.g., NF-κB) to promote expression of cytokines, chemokines and other activation markers.
TLR2 is a TLR that binds primarily to potential pathogens such as Gram positive bacteria, Mycobacteria, Borrelia (spirochetes), and yeast. TLR2 is present on the surfaces of certain host innate immune system cells, including myeloid lineage hematopoietic cells, as part of a heterodimer that may occur (in association with TLR1) as a TLR1/2 heterodimer, or (in association with TLR6) as a TLR2/6 heterodimer. Some innate immune system cells, including myeloid lineage hematopoietic cells, also express TLR4, which binds Gram negative bacteria and also recognizes LPS and lipoteichoic acid. TLR4 occurs as a cell surface (TLR4/4) homodimer. The bacterial components recognized in binding interactions by TLR2 and TLR4 include peptidoglycan, lipoteichoic acid, and tripalmitoylated lipoproteins. Interference with TLR2/6 heterodimer assembly impaired TLR2-mediated sepsis without affecting TLR4-mediated sepsis (Fink et al., 2013 J. lmmunol. 190:6410); hence, TLR2-mediated sepsis and TLR4-mediated sepsis can proceed via mechanisms that are independent of one another.
The pathophysiology of sepsis is complex but begins at a site of infection when a pathogen causes tissue injury, after first stimulating the innate immune system via TLRs and other PRRs, and subsequently provoking a response by the adaptive (acquired) immune system. Besides the pathogen-derived components (PAMPs) that are locally released early in this sequence of inflammatory events, accompanying tissue damage may also lead to the release of host cellular components that activate the innate immune response and are referred to as “danger associated molecular patterns”, or DAMPs. Examples of DAMPs include heat shock proteins (hsp) and alarmins, such as human mobility box group-1 protein. Coagulation factors, complement factors, mast cells, and platelets may all also play a role in this initial phase of localized tissue injury, which can lead to a local inflammatory response.
The hallmarks of inflammation include one or more of heat, redness, swelling and pain to which vasodilators are significant contributors, as mediated, for example, by bradykinins and histamine. The inflammatory response also involves the release of chemokines and activated complement factors that influence migration of neutrophils into the area of tissue injury and/or infection. Phagocytosis is enhanced by the interaction between PAMPs and PRRs on the surfaces of innate immune cells, with the subsequent release by these cells of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), and interferon-gamma (IFNγ). The primary purpose of the localized inflammatory process is to contain and destroy the pathogen. However if containment is not achieved and the pathogen is not eradicated, then the inflammatory process and the infection may spread. Once these events become systemic, the patient is at risk of developing sepsis (Hotchkiss et al., 2003 New Engl. J. Med. 348(2):138-150).
In systemic inflammation, the area of tissue injury that had previously been contained to a local milieu now instead sheds necrotic cellular components, pathogen and pathogen-derived debris, alarmins, PAMPs, and inflammatory cytokines into the circulation. The inflammatory process thus activates systemically many of the same innate immune system (e.g., PAMP-, DAMP-and PRR-driven) processes that were once localized. Global inflammatory phenomena characterize the resulting response, which includes systemic vasodilation, reduced vascular resistance, and increased cardiac output with tachycardia and tachypnea. The edema caused by vasodilation causes hypotension and hypoperfusion of critical organs. Ischemia in the organs promotes further tissue damage, which in turn further stimulates the inflammatory process. Concurrent with such widespread inflammatory events in sepsis, there is also evidence of immune suppression (manifest as lymphocyte anergy and the apoptosis-induced loss of CD4 T cells, B cells, and dendritic cells), along with reversible suspension of cellular functions in the organs (cell hibernation and cell stunning). These events contribute to secondary and nosocomial infections, complicating the host's status and further stimulating the inflammatory process (Fry, 2012, The American Surgeon 78:1-8).
Clinical trials of treating sepsis with various anti-inflammatory products such as corticosteroids, anti-endotoxin antibodies, anti-TNFα products, and IL-1 receptor antagonists, have been conducted without success (Hotchkiss et al., 2003 New Engl. J. Med. 348(2):138-150).
In a murine sepsis model, mice deficient for TLR2 or TLR2-derived signals were resistant to sepsis (Meng et al., 2004 J. Clin. Invest. 113(10):1473-1481). Meng et al. (2004) described a monoclonal antibody specific for the murine TLR2 extracellular domain that was able to block early events in a sepsis model system by interfering with the binding of a TLR2 agonist (P3CSK4, a mimetic of tripalmitoylated proteins found on bacterial surfaces) to TLR2.
Although the monoclonal antibody of Meng cross-reacts with human TLR2 (see also U.S. Pat. No. 8,623,353), this antibody is a murine immunoglobulin and requires specific binding interactions contributed by the immunoglobulin variable regions of two polypeptides (i.e., both the heavy and light chains). As such, problems associated with production of a protein macromolecule having six defined murine complementarity determining regions (CDRs) that originate in two distinct polypeptides (CDRs 1-3 from each of the immunoglobulin heavy and light chains) may hinder the development of a human therapeutic, such as proper folding and assembly of the variable regions, and removal of immunogenicity by engineering out murine determinants without compromising antigen binding specificity. Additionally, the rodent model may be limited in its predictive value for human sepsis, as noted by Decker (2004 J. Clin. Invest. 113:1387-1389).
A potential role for TLR2 in sepsis was also the subject of a report by Navarini et al. (2009 Proc. Nat. Acad. Sci. USA 106:7107-7112). These authors described experiments in which the TLR2 ligand Pam2Cys (5-(2,3-bis(palmitoyloxy)propyl)cysteine), a Mycoplasma-derived lipopeptide, elicited sepsis-like innate immune activation in a murine model of Listeria monocytogenes-driven neutrophil exhaustion. Co-administration to immunologically intact mice of an otherwise non-lethal (low) dose of Listeria along with Pam2Cys resulted in overwhelmingly lethal bacterial infections. Autopsy revealed depletion of neutrophils from bone marrow reservoirs (due to neutrophil migration) and the absence (due to neutrophil apoptosis) of live neutrophil infiltrates from tissue sites which, in experimental control animals that did not receive Pam2Cys, were abundant in neutrophils. Mice genetically deficient for TLR2 (tlr2−/−), by contrast, were free of disease when subject to the same inoculation regimen, a result the authors attributed to their lack of TLR2 receptors through which Pam2Cys could initiate the inflammatory cascade.
Use of a TLR2 ligand, Pam2Cys, thus detrimentally escalated innate immune activation that was instigated by a sub-lethal infection, causing it to progress to sepsis in much the same way as a lethal (high) Listeria dose: Neutrophils were driven to apoptosis without any apparent ability to forestall the immunosuppressive cytokine profile elaborated by phagocytes (macrophages and dendritic cells) involved in their clearance, thereby favoring an overwhelming bacterial infection (Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106: 6889-6890). Multiple additional reports describe significant neutrophil roles in the pathogenesis of sepsis, including exacerbation of disease by TLR2 activation (Navarini et al., 2009 Proc. Nat. Acad. Sci. USA 106:7107; Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106:6889; Alves-Filho et al., 2009 Proc. Nat. Acad. Sci. USA 106:4018; Zou et al., 2011 Shock 36:370; Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene et al., 2009 Infect. Immun. 77(12):5651).
Mice deficient for TLR4 or TLR4-derived signals were also sepsis-resistant, including experimental animals treated with an anti-TLR antibody (Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106(7):2348) and animals that were genetically engineered to lack TLR2 and TLR4 (Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene et al., 2009 Infect. Immun. 77(12):5651). The TLR4 antagonist Eritoran, however, failed to demonstrate efficacy for treating sepsis in a recent clinical study (Opal et al., 2013 J. Amer. Med. Assoc. 309(11):1154) despite previous encouraging reports for this agent (Tidswell et al., 2010 Crit. Care Med. 38:72; Barochia et al., 2011 Expert Opin. Drug Metab. Toxicol. 7(4):479), and the anti-TLR4 antibody described by Roger et al. (2009) had no effect on TLR2-mediated sepsis.
Despite previous work pointing to exacerbation of sepsis that results from activating TLR2 and/or TLR4 (Navarini et al., 2009 Proc. Nat. Acad. Sci. USA 106:7107; Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106:6889; Alves-Filho et al., 2009 Proc. Nat. Acad. Sci. USA 106:4018; Zou et al., 2011 Shock 36:370; Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene et al., 2009 Infect. Immun. 77(12):5651), efforts in the art to date have failed to arrive at an effective TLR2-and TLR4-directed immunomodulator for sepsis (Lorne et al., 2010 Intensive Care Med. 36:1826) that has a beneficial mechanism of action and favorable efficacy profile, and that further offers ease of manufacture and is nonimmunogenic in humans.
Clearly there remains a need for improved compositions and methods to treat, reduce the severity of, or reduce the likelihood of occurrence of TLR2-and/or TLR4-mediated sepsis. Certain of the presently disclosed invention embodiments address this need and provide other related advantages.
Cancer
Cancer remains a devastating and largely intractable disease with significant unmet needs in the areas of patient treatment, clinical outcome and overall survival. The American Cancer Society (ACS) estimates that there will be over 1.6 million new cases of cancer diagnosed in the United States in 2012, not including non-invasive carcinoma in situ and also not including new cases of basal cell carcinoma and squamous cell skin cancer. The ACS also projects that there will be 577,190 cancer-related deaths in 2012, or an average of 1500 Americans dying each day from cancer, making cancer the second most common cause of death in the U.S., after heart disease. In 2007 the total medical cost of cancer in the U.S. was $226.8 billion, including direct medical costs for treatment of $103.8 billion and indirect costs due to lost productivity and premature death of $123 billion. The mean five-year survival rate overall for U.S. cancer patients has improved from 49% in 1975-1977 to 67% in 2001-2007. There clearly, however, still remains a pressing need for improved treatments and enhanced overall outcomes for cancer patients.
Transplantation
Organ transplantation is often the best or only treatment option for end-stage organ failure, such as kidney disease, chronic conditions such as severe cirrhosis of the liver, and cancer, such as liver cancer, leukemias and lymphomas. Both solid organ and bone marrow transplants are performed in order to treat patients in need. It is estimated that about 100,000 solid organ transplants were performed worldwide in 2007, and of the roughly 30,000 bone marrow transplants performed annually, about 15,000 are allogenic transplants. Kidney, liver and heart transplants are the most common solid organ transplants.
The worldwide demand for donor tissues and organs far surpasses the supply, and there is a significant need in transplantation medicine to improve the availability of donor organs and minimize the long term risk of rejection. For example, the World Health Organization (WHO) has estimated that only 10% of those in need of kidney transplants manage to get one. The National Kidney Foundation claims that about 18 patients die daily while waiting for a transplant of a vital organ such as a heart, liver, kidney, pancreas, lung, or bone marrow. Furthermore the long-term graft survival rates for kidney and liver transplants are about 60-70% at 5-10 years post-transplant.
Rejection of the donor tissue by the host immune system is one of the largest problems faced in allogenic transplants. Despite donor-recipient human leukocyte antigen (HLA) histocompatibility matching and ABO blood group testing to provide matched donor tissue to the recipient, patients receiving transplants must undergo immunosuppressive treatment in an effort to prevent graft rejection or, in the case of bone marrow transplants, graft-versus-host disease (GVHD). Most immunosuppressors target T cells or cytokines secreted by T cells, and types of immunosuppressive agents currently used include monoclonal antibodies to lymphocytes and cytokine receptors (e.g., anti-IL-2Rα), calcineurin inhibitors (e.g., cyclosporine and tacrolimus), and cytokine receptor signal transduction inhibitors (e.g., sirolimus) (Chinen and Buckley, J Allergy Clin lmmunol, 2010, 125(2 Suppl 2):S324-S335). The downside to using immunosuppressive agents, sometimes over a course of several months, is that while protecting the graft from being rejected by the host immune system, or vice versa in the case of GVHD, they make the recipient especially vulnerable to infections and malignancies.
In addition, despite advances in immunosuppressive treatments which have significantly improved first year graft survival, long-term survival is still unsatisfactory. In a study of long term kidney graft survival, Fernandex-Rodriguez et al (Transplantation Proceedings 2009 41(6): 2357-2359) followed 1,029 first renal transplantations performed between November 1979 and December 2007, observed renal graft survival at 1, 5 and 10 years and correlated the results to the immunosuppressive therapy used, including azathioprine (AZA), cyclosporine (CsA), and tacrolimus (TAC). The findings indicated that graft survival rates at 5 and 10 years post-transplant were, respectively, 56% and 46% on AZA, 69% and 54% on CsA, and 77% and 60% on TAC. The study concluded that despite the decrease in acute rejection in kidney transplants, there was a significant decrease in renal graft survival after 12 months. Another study by Ruiz et al (Arch Surg 2006 141:735-742) reported liver graft survival at 1, 3 and 5 years post-transplant of 70%, 65% and 65%, respectively, and kidney graft survival at 1, 3 and 5 years post-transplant of 76%, 72% and 70%, respectively, thereby demonstrating that there is a significant decrease in graft survival following the first year.
As a semi-allograft, the maternal-host acceptance and tolerance of an embryo and placenta is similar in many ways to an allogeneic organ transplant. Implantation of the blastocyst into the uterine wall is a critical checkpoint for a successful pregnancy and results in the embryo adhering to the uterine lining and generating a vascular connection. Implantation failure can result in repeated miscarriages and failed in vitro fertilization (IVF) attempts. In particular, embryo implantation success rates in IVF patients vary widely, and success rates ranging from about 15% to about 30% have been reported (see, e.g., Croo et al. Human Reproduction, 15(6):1383-1388, 2000). Inflammation and the mother's immune response are believed to play a role in the successful implantation of an embryo.
Inflammation also plays a role later on in pregnancy, such as in preeclampsia and eclampsia, which affect an estimated 5-8% of all pregnancies and are the leading cause of maternal and fetal illness and mortality worldwide. Preeclampsia is characterized by high blood pressure and proteinuria, and if left unchecked, it can lead to the seizures of eclampsia. During pregnancy, the maternal adaptive immune response is down-regulated, and the innate immune response is enhanced. However, the innate immunity also must be regulated, and it has been shown that neutrophils, non-antigen specific white blood cells of hematopoietic origin that are typically associated with inflammatory and anti-microbial responses, play a large role in preeclampsia (Cadden and Walsh, Hypertens Pregnancy (2008) 27(4):396-405).
Indeed, studies indicate that innate immune cells, such as neutrophils (often referred to as polymorphonuclear neutrophils, or PMN), are important in shaping, enhancing and regulating the adaptive immune response (see, e.g., Soehnlein, Trends in Immunology, 2009, 30(11):511-512 and Muller et al, Trends in Immunology, 2009, 30(11):522-530). Recent studies indicate that neutrophils may play a significant role in antitumor reactions (DiCarlo et al., 2001 Blood 97:339; Mumm et al., 2011 Canc. Cell 20:781) and in transplant rejection, in addition to the historical role of lymphocytes. In particular, Soo et al. (J. Heart and Lung Transplantation, 2009, 28(11):1198-1205) examined pre-operative neutrophil adhesion molecule expression after in vitro stimulation with LPS or PMA and then correlated these results with actual allograft success. Interestingly, pre-operative neutrophil surface CD11 b expression after LPS stimulation correlated proportionally with the degree of rejection as detected in the first endomyocardial biopsy sample post heart transplantation, and the authors concluded “that neutrophils may contribute more to cardiac allograft rejection than previously thought.”
Another study identified increased IL-17 and neutrophilia in the broncho-alveolar lavage of patients undergoing acute lung transplant rejection (Chest 2007 131(6):1988-9). Furthermore, neutrophils have been shown to play a significant role in xenotransplantation rejection (Transplant Immunology 2009 21:70-74, Transplantation 2004 78: 1721-1718), and means of attenuating neutrophil activity may play a significant role in improving the success of this type of transplantation approach. There is also increasing evidence that neutrophils can be activated to express MHC Class II molecules and develop antigen presenting cell (APC) characteristics and release cytokines such as IL-4, IL6, IL-10, IL-12, and TNFa and suppress T cell activity (e.g., Muller et al., 2009 Trends in Immunology 30(11):522-530, 2009; Vasconcelos et al., 2006 Blood 107:2192-2199; Rodriguez et al., 2009 Cancer Research 69:1553-1560, 2009. Therefore the role of neutrophils may also be involved in allograft and xenograft acceptance.
Neutrophils are clearly of interest for research due to their role in inflammation and infection as well as other immune functions, including roles in pregnancy, transplantation and autoimmunity. A number of cell surface markers present on neutrophils have been utilized in an effort to characterize, identify and determine their activation state, such as CD64, CD11b, and CD83; however few neutrophil markers exist that can readily be used to identify a distinct subset of neutrophils in the same way that other hematopoietic cell surface markers can be used to categorize adaptive immune cells, for instance, according to maturational state, differentiation lineage, and functional properties (e.g., CD4 versus CD8 T lymphocytes, surface immunoglobulin (sig) changes in B lymphocyte differentiation and maturation, and other regulatory cell markers, e.g., CD45 isoforms, distinct integrin α and β chain heterodimer expression, etc.). Accordingly, a need exists for more refined neutrophil markers in order to functionally characterize these cells. (See, e.g., Mason et al., (Eds.), Leukocyte Typing VII, 2002 Oxford Univ. Press, USA.)
An increased understanding of the mechanisms resulting in graft rejection have lead to advancements in the availability and mechanistic understanding of immunosuppressants; however, there is still a significant medical need for immunosuppressive agents that are capable of improving the acceptance of donor organs and minimizing the risk of rejection while at the same time placing recipients at a lesser risk of infections and malignancies. In particular, there is an unmet need for treatments that improve long term graft survival and also improve the success of xenotransplantation. In addition, immunosuppressive agents that are useful for conception, providing improved rates of implantation and a reduced risk of multiples, as well as preventing conditions like preeclampsia, are needed.
The compositions and methods of the present invention address the needs described above and offer other related advantages.